Life Sciences, Vol . 24, pp . 809-816 Printed in the U .S .A .

Pergamon Press

HUNTINGTON'S DISEASE AND ITS ANIMAL MODEL : ALTERATIONS IN KAINIC ACID BINDING Kevin Beawnont, Yves Maurln, Terry D . Reisine, Jeremy Z . Fields Ernest Spokes*, Edward D . Bird* and Henry I . Yamamura** Department of Pharmacology, University of Arizona Health Sciences Center, Tucson, Arizona 85724 ; and*Addenbrookes Hospital, Cambridge, England . (Received in final form January 24, 1979) SUMMARY The density of 3 H-kainic acid (KA) binding was determined in several regions of Huntington's Diseased (HD) and control human brains . 3 H-Kainic acid binding was significantly reduced by 55X in the cau date nucleus and .by 53~ in the putamen of HD brains . In addition, 9 H-KA binding was determined in rat striatum at various intervals following lesion with KA, a procedure which produces an animal model of HD . After KA lesion, 3 H-KA binding in the rat striatum underwent a slow reduction, reaching 25~ of control after 6 weeks . Several properties of 3 H-KA binding to rat brain membranes were also investigated, including inhibition by ions, regional distribution and displacement by various compounds . The findings confirm the validity of the KA-lesioned model for HD and suggest a post-synaptic location for kainic acid receptors in the striatum . INTRODUCTION Kainic acid (KA), a cyclic analogue of L-glutamic acid, is an extremely potent neuronal depolarizing agent (1) . Upon injection into the rat striatum, KA causes the degeneration of neuronal cell bodies located at the site of injection, while axons passing through or terminating in the area of injection are unaffected (2) . The neurotoxic effects of KA may be the result of excessive neuronal depolarization (3) mediated by synaptic glutamate receptors . A synaptic site of action is supported by studies demonstrating that binding of radiolabeled KA to rat brain is of high affinity, saturable, displaceable by L-glutamate, stereospecific, concentrated in synaptic membranes, and unevenly distributed in rat brain regions (4) . Histological and neurochemical alterations resulting from striatal injection of KA (2,5,6) or L-glutamic acid (6 ,7) are remarkable similar to those occurring in Huntfington's Disease (HD), a hereditary disorder characterized behaviorally by choreic movements and dementia and pathologically by shrinkage of the caudate nucleus and putamen, accompanied by neuronal degeneration and gliosis (8) . Present addresses : KB : Department of Neuropharmacology, Synthelabo-LERS, Bagneux, France . YM : Laboratoire de Neurochimie, INSERM U 134, Hopital de la Salpetriere, Paris, France . JZF : Department of Pharmacology, Chicago Medical School, Chicago, I11 . EDB : Department of Neuropathology, McLean Hospital, Boston, Mass . to whom reprint requests should be sent 0024-3205/79/090809-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd

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Consequently, striatal KA-induced degeneration serves as a model for investigation of the etiology and therapy of HD (2) . We report here on the density of the KA binding site in HD and control human brain, and on the alterations of KA binding produced by striatal KA lesions . METHODS AND MATERIALS Tissue : Two ug of KA in 1 ul saline were stereotaxically injected into the striatum of pentobarbital-anesthetized adult male Sprague-Dawley rats (200-250 gms) . Coordinates for the injection, determined using the atlas of Konig b Klippel (9), were 7 .8 mm anterior and 2 .6 mm lateral from the interaural line and 4 .8 mm vertically from the surface of the brain . The solution was injected through a 30-gauge needle over a period of 3 minutes, and the needle was left in place for an additional 3 minutes before withdrawal . At various times after lesioning, animals were sacrificed by decapitation, and left and right striata were dissected and frozen . Pathological specimens of human brains were obtained from 16 patients with HD, ages 42-74 years, (mean = 59 years) and from 18 control individuals without infectious or malignant disease of the central nervous system, ages 17-88 years (mean = 57 years) . HD patients were undergoing therapy with neuroleptics, diazepam, and morphine at time of death . Tissue was frozen at -70°C until the day of the assay . Both human and animal tissues were throughly washed by centrifuging and resuspending four times in approximately 150 volumes of fresh buffer prior to assay of 3H-KA binding . For studies involving displacement of 3H-KA by compounds or by ions, whole rat brain synaptic membranes were used . Synaptic membranes were prepared by the method of Zukin et al . (10) and were then frozen for a period of 1-7 days . Synaptic membranes were thawed and washed four times by centrifuging and resuspending in fresh buffer prior to use . 3H-KA binding : 3H-KA binding was determined by a modification of the method described by Simon, et al . (4) . Throughly washed tissue homogenates (0 .3 - 0 .6 mg protein/assay) were incubated in triplicate with 0 .5 - 150 nM 3H-KA (2 .3 Ci/ mmole, New England Nuclear) in 4 ml of 0 .05 M tris-citrate buffer, pH 7 .1 . After 30 minutes of incubation at 4°C, membranes were sedimented by centrifuging for 10 minutes at 48,000 x g . The pellets so obtained were rapidly surfacewashed twice with 5 ml of ice cold distilled HZO and solubilized with NCS (Amersham/Searle) . Toluene-omnifluor scintillation cocktail was added and radioactivity determined by liquid scintillation spectrometry . Non-specific binding was determined in the presence of 1 mM L-glutamic acid and subtracted from the total bound to obtain specific binding . Protein concentrations were determined by the method of Lowry et al . (11) . Choline acetyltransferase (CAT) activity : 5 ul of unwashed tissue homogenate (3 .3% in 50 mM NaKPOy buffer) were added to 25 u l of an assay mixture containing 0 .4 ml Na HPOg/NaH 2 PO buffer (0 .2 M, pH 7 .4), 0 .17 ml eserine salicylate (0 .001 M), 0 .~6 m7 MgC7 2 ~0 .1 M), 0 .12 ml NaCI (3 M), 0 .12 ml choline chloride (0 .02 M), and 0 .1 ml 14 C-acetyl CoA (0 .02 mCi/ml) . After 20 minutes incubation at 37°C, 0 .1 ml of tetraphenylboron in 3-heptanone (50 mg/ml) was added and the mixture was cooled in an ice bath for 5 minutes, then centrifuged for 2 minutes in a Beckman Microfuge B . The radioactivity (3H-acetylcholine fornied) in 50 ul of the organic layer was determined by liquid scintillation spectrometry . RESULTS Scatchard analysis of saturation isotherms for 3H-KA binding to washed whole rat brain homogenates revealed a binding site with a dissociation constant (Kp) of 5 .3 t 1 .8 nM and a receptor density (Bmax) of 182 ± 47 fmol/mg protein (n=5) . The density of 3H-KA binding at .a concentration of 10 nM varled among regions of rat brain, with the greatest density in the striatu~hippocampus> cerebellum ~ cerebral cortex>midbrain = pons-medulla . Several neuroexcitatory amino acids displaced 3H-KA from whole rat brain homogenates (Table 1) .

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Kainic Acid Binding in Huntington's Disease

TABLE I COMPOUND KAINIC ACID QUISQUALIC ACID DIHYDROKAINIC ACID

IC so (MICROMOLAR) 0 .006 0 .032 5

L-GLUTAMIC ACID D-GLUTAMIC ACID DL-HOMOCYSTEIC ACID L-CYSTEIC ACID L-HOMOCYSTEIC ACID L-CYSTEINE SULFINIC ACID

0 .21 12 13 16 18 18

L-GLUTAMINE

13

DL-a-AMINOADIPIC ACID D-ASPARTIC ACID L-ASPARTIC ACID N-METHYL-D-ASPARTIC ACID

120 400 400 850

Inhibition of 3 H-KA binding to rat brain synaptic membranes . Values represent the concentration of the compound which inhibits by 50~ the specific binding of 5 nM 3 H-KA to whole rat brain synaptic membranes and are the means of 2-4 experiments . Several compounds produced little or no inhibition at a concentration of 100 uM, including GABA, muscimol, bicuculline, strychnine, glycine,taurine . histamine . adenosine . dopamine, atropine sulfate, serotonin, naltrexone, L-carnosine, L-cysteine, L-histamine, hemicholanium-3, sodium Phenobarbital, diazepam, metrazol, diphenylhydantion, clozapine, amitriptyline, theo.phylline, ouabain, 2,4dinitrophenol, pyridoxal-5'-P0, 2-mercaptoethanol, dithiothreitol, caffeine, ATP, AMP, GMIP, uradine monophosphate, inosine-5'-PO y , inosine, cytosine, guanosine, L-ascorbic acid, 6-hydroxydopamine, 6-hydroxyDOPA, o-phospho-L-serine, DL-a-aminopimelic acid, and Y-hydroxybutyri c aci d . Half-maximal displacement (IC SO ) of the specific binding of 5 nM 3H-KA occurred with 0 .006 pM unlabeled KA, 0 .21 uM L-glutamate, and 12 uM D-glutamate . However, L-aspartac acid, D-aspartic acid, and N-methyl-D-aspartic acid were considerable less potent, IC o's being greater than 0 .1 mM for all three compounds . Several inhibitors of L-g~utamate-induced excitations were relatively ineffecThus, the ICso for distive at inhibiting 3 H-KA binding in our assay system . placing 5 nM 3 H-KA was greater than 0 .1 mM for a-methyl-DL-glutamic acid, Lglutamic acid diethylester (L-GDEE), 2-amino-4-phosphonobutyric acid, and the glutamate synthetase inhibitor L-methionine-DL-sulfoxa mane, as well as for the aspartate-antagonist (12), DL-a-aminoadipic acid . Several compounds produced little or no effect at a concentration of 0 .1 mM upon 3 H-KA binding to rat brain synaptic membranes (Table 1, legend) . The effects of several ions upon 3 H-KA bindin~ to whole rat brain synaptic membranes were determined . C1 - does not inhibit H-KA binding, since increasing the concentration of tris-C1 buffer up to 200 mM does not inhibit 3 H-KA binding . Therefore, the inhibition produced by a cation with C1 - as its counterion is probably due solely to the cation . Monovalent cations, tested with C1 - as the counterion, inhibit 3 H-KA binding in a dose-dependent manner . The inhibitory potency of the alkali cations decreases with increasing molecular weight, Li+

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being the strongest inhibitor and Cs+ the weakest inhibitor (Table 2) . Na + has an ICsp of 61 mM when tested with C1 - as the counterion, which is similar to the ICsp's of 50 mM and 66 mM obtained with Br - and I- , respectively, as counterlons . The divalent can ons Ca m, Mn~, aüd Mgr are considerably more potent inhibitors of 3H-KA binding than are the monovalent canons (Table 2) . TABLE II ION

(mMg

ION

(mMg

LiCI NaCI KC1 NH,, C1 RbCI CsCI

32 61 149 171 192 197

CaC1 2 MnC1 2 MgC1 2

2 .3 2 .8 '24

NaBr NaI

50 66

Inhibition of 3H-KA binding to rat brain membranes by ions . Values represent the concentration of ion necessary to inhibit by 50% the binding of 5 nM 3H-KA to whole rat brain synaptic membranes . For experiments involving divalent cations, this -~HC1 buffer was used rather than tris-citrate, in order to avoid chelation of divalent cations by citrate . Substitution of tris -HC1 buffer did not appreciably alter control binding values or binding data for monovalent cations . ICsp's were determined by log-probit analysis of the mean values from 2 to 5 experiments . 3H-KA binding site density and CAT activity in individual rat striate were determined at various intervals after striatal lesion with KA (Figure 1) . The striatum contralateral to the lesion served as control for each animal . In accordance with previous reports (5,6) CAT activity of lesioned striatum was reduced to 30% of (control = 185 t 10 nmole/mg protein/hr) within 5 days of lesioning . In contrast, 3H-KA binding density was not significantly different from control at 5 or 8 days after lesioning . However, 3H-KA binding was reduced to 78% of control after 14 days, decreasing to 25% of control at 48 days after lesion . Preliminary studies indicated that kinetic values for 3H-KA binding to thoroughly washed control hupen cerebral cortex (Kp - 7 nM, Bmax = 118 fmole/mg protein) and hupen cerebellum (Kp = 11 nM, Bmax - 145 fmole/mg protein) are similar to kinetic values obtained with whole rat brain . In addition, the potenc .y of L-gl utamate 1 n di s pl aci ng s H-KA from hupen cerebral cortex (ICsp 0 .2 uM) and hupen cerebellum (ICsp - 0 .6 uM) is similar to its potency in displacing 3 H-KA from whole rat brain membranes (ICS p - 0 .21 uM) . The density of 3 H-KA binding was determined in human brain regions and found to be significantly decreased in the caudate nucleus and putamen of H .D . brains as compared to control human brains (Figure 2) . 3 H-KA binding density was significantly reduced by 55% in H .D . caudate nucleus, from a mean of 115 .5 ± 7 .7 fmole/mg protein in control caudate nuclei (N - 12) to 51 .5 t 8 .2 fnale/mg protein in H .D . caudate nuclei (N = 11), (pc0 .001 by two-tailed, unpaired "t" test) . The density of 3 H-KA binding was significantly reduced by 53% in H .D . putamen, from 128 .5 ± 8 .8 fmole/mg protein in control utamen (N - 12) to 60 .0 ± 5 .8 finol e/mg protein i n H . D. putamen (N - 11) , (p pc 0 .001 by two-tai 1 ed "t" test) . 3H-KA binding was not significantly correlated with age at death, for either HD or control individuals, and was not correlated with duration of H .D . symptoms . In cerebellum (N s 3), frontal cortex (N = 7), and globus pallidus (N = 5), the density of 3H-KA binding in HD brains did not differ significantly from controls (data not shown) .

1Cainic Acid Binding in Huntington's Disease

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100

75

1

3 H-KAINIC ACID &ND~Iß

1 1 1 1

O H z

1 1 1 1 1 1 _I

813

50

O

1

1

11____ _ ~~ CAT ACTIVRY

25

10

20

30

DAYS

AFTER LESION

40

50

FIGURE 1 Time course of alterations of 3 H-KA binding and CAT activity of rat striatum following KA lesion . For each animal, values for 3H-KA binding (finales/mg protein) and CAT activity (males acetylcholine synthesized/mg protein/hour) were determined for the lesioned striatum and compared to the contralateral control striatum . Each point represents the mean t S .E .M . for 4-10 animals . DISCUSSION We have found a significant decrease in 3 H-KA binding in both HD and KAlesloned striatum . In our hands, the dissociation constant of the rat brain bindiny site for 3 H-KA is 5 nM, which is a 10-fold higher affinity than that (59 nM) reported previously by Simon, et al . (4) . This discrepancy may be due to the more extensive washing procedure that we utilized to remove the large quantities of endogenous glutamate, ca n ons, and other possible unknown inhibitors of 3 H-KA binding present in brain tissue . In support of this possibility we found that aliquots of the supernatants of the first three tissue washes significantly inhibit 3 H-KA binding to thoroughly (4X) washed brain tissue (data not shown) . The regional distribution of 3 H-KA binding in rat brain by our determination is quite similar to that reported by Simon et al (4), which indicates that we are probably measuring the same binding site . We have confirmed previous findings (4) that the ability of certain amino acids to produce neuroexcitation does not clearly parallel their potency in displacing 3 H-KA binding . In particular, the lack of affinity of L- and D aspartate and of N-methyl-D-aspartate for the 3 H-KA binding site contrasts with

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PUTIUAEN

Vol . 24, No . 9, 1979

CAUDATE NUCLEUS

100

100

"~S Oo

CONTIIOL

HD

CONTROL

ND

FI GORE 2 Density of 3H-KA binding in the caudate nucleus and putamen of HD and control human brains . Specific 3H-KA binding was determined at a concentration of 10 nM, as described in Materials and Methods . Each point represents a different brain . Bars represent the mean value for each area . their high neuroexcitatory potencies . This finding is consistent with neurophysiological evidence that "aspartate-preferring" receptors are distinct from "glutamate-preferring" receptors (12,13), the latter having a greater sensitivity to KA . Several amino acids with widely varying potencies in exciting mammalian central neurons (D-glutamic acid, DL-homocysteic acid, L-cysteic acid, L-cysteine sulfinic acid) as well as L-glutamate, which is without neuroexcitatory effects in the cat spinal cord (14,15), have similar affinities for the 3H-KA binding site, all yielding ICso s within the range of 10-20 uM . The affinity of these neuroexcitatory amino acids for the 3 H-KA binding site is 100fold less than the affinity of KA itself . Therefore, the site mediating KAinduced neuroexcitation most likely represents only one of several receptors mediating amino acid-induced excitations . The low potency of the glutamate antagonists L-GDEE, a-methyl-DL-glutamate, and 2-amino-4-phosphonobutyric acid at displacing 3H-KA may indicate that 1) these antagonists act elsewhere than the amino acid recognition site to block neuronal excitation, 2) antagonists bind to a different conforTnation of receptors than do agonists, or 3) KA acts at a subpopulation of excitatory receptors that is not sensitive to these inhibitors . This third possibility is supported by the recent finding that KAinduced depolarization of rat thalamic neurons is not blocked by concentrations

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Kainic Acid Binding in Huntington's Disease

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of L-GDEE which are effective against L-glutamate-induced depolarization (16) . The discrepancy between the neuroexcitatory potency of several compounds and their ability to displace 3H-KA from brain membranes raises the possibility that the 3H-KA binding site measured in vitro does not correspond to the receptor that mediates KA-induced neuroexcitation in vivo . However, the findings of Simon et al . (4) that the binding site is localized exclusively in neuronal tissue and is most concentrated in synaptic membranes strongly indicates that the binding site is relevant to the neuroexcitatory actions of KA . Furthermore, we have found that the close analogue dihydrokainic acid, which is a very weak neuronal excitant (13), has a 1000-fold lower affinity for the 3H-KA binding site than does KA . This finding also indicates that under these conditions 3HKA is not binding to a neuronal L-glutamate uptake site, since dihydrokainic acid is a more potent inhibitor of L-glutamate uptake into rat striatal synaptosomes than is KA, which is itself very weak in this respect (17) . The likelihood that the 3H-KA binding site measured in vitro corresponds to the site mediating toxicity in vivo is also supported by t~studies of Campochiaro and Coyle (18) which demonstrate that during the maturation of the rat striatun, the increase in susceptibility to KA neurotoxicity correlates temporally with the increase in 3H-KA binding . Several ca n ons inhibit 3H-KA binding, some at physiological concentrations . Divalent ca n ons are more potent than monovalent ca tions, although the inhibition by divalent can ons may be partially attributable to complex formation with KA . At their IC , Na and Mn~ produced a lowering in affinity with no than e in Bmax of 3H-~ binding to rat brain synaptic membranes (unpublished data . Thus, although the affinity of KA for its receptor in vitro is in the nanomolar range, considerably higher concentrations may be required to produce half-maximal binding in vivo due to competitive inhibition by ca n ons . The relatively slow decrease in 3 H-KA binding following KA lesion was unexpected since dendrites and cell bodies, which would be expected to bear the glutamate receptors, are destroyed within 2-3 days of lesioning (19) . However, Olney and de Gubareff (20) have recently reported that postsynaptic densities remain adhering to the presynaptic terminals at 21 days after KA lesion . The postsynaptic densities are thought to contain receptors for neurotransmitters, and their continued presence after dendrites and cell bodies have degenerated may account for the relatively slow decrease in KA receptor density after lesion . The eventual 75% decrease in KA binding following lesion suggests that a majority of striatal KA receptors are located on neurons with cell bodies intrinsic to the striatum, possibly at sites post-synaptic to corticostriatal gl utamatergi c afferents . 3H-KA binding was found to be significantly reduced by 55% in the caudate nucleus and by 53% in the putamen of HD brains as compared to control human brains . This 3H-KA binding site measured in human brain is similar to the binding site of rat brain by several criteria, including dissociation constant, binding density, and affinity for L-glutamate . Drugs present in brain samples were most likely removed prior to assay by the thorough tissue washing procedure . In any case, representatives of each of the classes of drugs used by HD patients do not significantly effect 3H-KA binding at a concentration of 100 u M . The finding that 3 H-KA binding is decreased in HD caudate and putamen but not in frontal cortex, cerebellum, or globus pallidus provides a preliminary indication that generalized destruction of KA sensitive sites, which may be postsynaptic to glutamate-releasing neurons, does not occur in this disease . In addition, the reduced binding of 3H-KA in HD and KA-lesioned striatum supports the validity of the KA-lesion animal model for HD . ACKNOWLEDGEMENTS We would like to acknowledge the technical assistance of David Chapman and Thomas McManus and the secretarial assistance of Cathy Kousen . We thank Dr . Ante Padjen for donating samples of quisqualic acid and Dr . Michael Schmidt for

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for providing samples of dihydrokainic acid . We would also like to thank Dr . Peter C . Johnson for providing histological verification of the lesions . REFERENCES 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .

Shinozaki, H . and S . Konishi,(1970), Brain Res . 24 : 368-371 . Coyle, J .T ., R . Schwarcz, J .P . Bennett, an~d~P . Câmpochiaro,(1977), P~roc .. in Neuro- psychopharmacol . 1 : 13-30 . 0~, J .W . F .~Rhee andO .L . Ho, (1974) Brain Res . _77 : 507-512 . Simon, J .R ., J .F . Contrera, and M .J . Kuhar, 97~j,J Neurochem . _26 : 141-147. Coyle, J .T . and R . Schwarcz, (1976) Nature Lond 2 3 : 517- 19 . l~-519 . McGeer, E .G . and P .L . McGeer, (1976) Naturé Olney, J .W . and T . de Gubareff, (1978 Nature 271 : (557-559) . Enna, S .J ., L .Z . Stern, G .J . Was tek and~I .~Yamamura,(1977), Life Sci . _20 : 205-212 . Konig, J .F .R . and R .A . Klippel,(1967), The Rat Brain , Robert E . Kruger Publishing Co ., Huntington, New York . Zukin, S .R ., A .B . Young, and S .H . Snyder, (1974) Proc . Nat . Acad . Sci . _71 : 4802-4807 . Lowry, O .H ., N .J . Rosebrough, A .L . Fair, and R .J . Randall,(1951), J . Biol . Chem . 193 : 265-275 . Biscoe, T .J . R .H . Evans, A .A . Francis, M .R . Martin, J .C . Watkins, J .Davles, and A . Dray,~1977), Nature 27ß : 743-745 . Johns ton, G .A .R ., D .R . Curtis, J . Davies, and R .M . McCulloch,(1974~ Nature 248 : 804-805 . Cûrtis, D .R . and J .C . Watkins, 1976 ), ~J . Ph,j!siol . 166 : 1-14 Curtis, D .R . and J .C . Watkins,~1960),J . Neurochem~ : 117-141 . Hall, J .G ., T .P . Hicks, and H . McLennan, 19 8 ,Neurôsci . Lett . 8 : 171-175 . - 0. Biziere, K . and J . T . Coyle,(1978),Neurosci . Letters : Campochiaro, P . and J .T . Coyle,(1978 Proc Nat . Acad . Sci . 75 : 2025-2029 . Hattori, Y . and E .G . McGeer,(197~, Bra n Res . Brain Res.24 : 368-371 . Olney, J .W . and T . de Gubareff,(197